Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, similar to 32 angstrom from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Cu2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Cu2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Qmutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (similar to 60%WTECAO TPQ), I342F/E573Qhas almost no amine oxidase activity (similar to 4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+ -substrate reduced TPQ(TPQ(AmQ)) CutTPQsemiquinone (TPQ(SQ)) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQ(SQ) or destabilizes Cu+-TPQ(SQ): Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state: Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.